Chromosomal DNA isolation from E. coli protocol
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Solutions/reagents:
- culture grown in your favorite medium
- ice-cold Killing Buffer
- TE
- 10% SDS
- 0.5M EDTA
- isopropanol
- <a name="RNase A">RNase A
<tab>(25mg/ml)</a> - <a name="proteinase K">proteinase K
<tab>(25mg/ml)</a> - ethanol
- 3M Na-Acetate
- distilled water
Equipment:
- Centrifuge
- Incubator
Steps:
- Measure out 1 volume ice-cold Killing Buffer into culture grown in your favorite medium.
Vortex the mixture for a few secs.
Store the tube on ice.
Samples should be processed as fast as possible. - Centrifuge at maximum speed for 3 mins at 4°C, gently aspirate out the supernatant and discard it.
- Measure out 300 µl of TE into Eppendorf tube (1).
Add 40 µl of 10% SDS.
Add 3 µl of 0.5M EDTA.
Resuspend pellet by vortexing/by shaking vigorously. - Incubate at 65°C for 5 mins.
- Add 750 µl of isopropanol.
Vortex the mixture for a few secs. - Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
- Add 500 µl of TE.
Add 2 µl of <a href="#RNase A" >RNase A</a>.
Resuspend pellet by vortexing/by shaking vigorously. - Incubate at 65°C for 30 mins.
- Add 2 µl of <a href="#proteinase K" >proteinase K</a>.
Incubate at 37°C for 15 mins. - phenol extract (2x phenol & 2x chlorophorm).
- Add 1 ml of ethanol.
Add 40 µl of 3M Na-Acetate.
Store at room temperature for 12 hrs(overnight). - Centrifuge at maximum speed for 15 mins at 4°C, gently aspirate out the supernatant and discard it.
Add 50 µl of distilled water.
Resuspend pellet by vortexing/by shaking vigorously.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 13 hrs, 13 mins
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